Indian Journal of Medical Microbiology, (2015) 33(1): 101-109 1

Original Article

Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from
wounds in a tertiary care hospital
MA Ansari*, HM Khan, AA Khan, SS Cameotra, MA Alzohairy

Abstract
Purpose: Different approaches have been used for preventing biofilm-related infections in health care settings. Many
of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant
strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis
infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology
may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study
was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance
S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red
Agar methods. Materials and Methods: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning
electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. Results: The minimum
inhibitory concentration (MIC) was found to be in the range of 11.25-45 μg/ml. The AgNPs coated surfaces effectively
restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect
bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides
matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the
glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as
low as 50 μg/ml. Conclusions: Our findings suggested that AgNPs can be exploited towards the development of potential
anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play
major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm.

Key words: Anti-biofilm, AgNPs, confocal laser scanning microscopy, exopolysachharide, scanning electron microscopy

Introduction of indwelling medical devices has had considerable

Staphylococci are most often associated with chronic
infections of implanted medical devices.[1] The use of
indwelling medical devices is important in the treatment
of critically and chronically ill patients, however, bacterial
colonisation of implanted foreign material can cause
major medical and economic sequel. The increased use
*Corresponding author (email: <azammicro@gmail.com>)
Nanotechnology and Antimicrobial Drug Resistance Research
Lab, Department of Microbiology (MAA, HMK), and Department
of Anatomy (AAK), Jawaharlal Nehru Medical College and
Hospital, Aligarh Muslim University, Aligarh, Institute of Microbial
Technology (IMTECH) (SSC), Sector 39-A, Chandigarh, Department
of Medical Laboratories College of Applied Medical Science
Buraydah Colleges (MAA), Buraydah 51452 Saudi Arabia
Received: 11-07-2013
Accepted: 29-01-2014
Access this article online
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PMID:
***
DOI:
10.4103/0255-0857.148402
impact on the role of staphylococci in clinical medicine.
The predominant species isolated in these infections are
Staphylococcus epidermidis and S. aureus, their major
pathogenic factor being ability to form biofilm on polymeric
surfaces.[2] Biofilm producing staphylococci frequently
colonise catheters and medical devices and may cause
foreign body-related infections. They easily get attached to
polymer surfaces.[3] Bacterial biofilms are a predominant
challenge to wound healing.[4]
The first recorded observation concerning biofilm
was probably given by Henrici in 1933, who observed
that water bacteria are not free floating but grow upon
submerged surfaces.[5] Biofilm consists of multilayered
cell clusters embedded in a matrix of extracellular
polysaccharide (slime), which facilitates the adherence
of these microorganisms to biomedical surfaces and
protect them from host immune system and anti-microbial
therapy.[6] Biofilm formation is regulated by expression of
polysaccharide intracellular adhesin (PIA), which mediates
cell to cell adhesion and is the gene product of icaADBC.[7]
Various reports attest to the presence of icaADBC gene in
S. aureus and S. epidermidis isolated from infections
associated with indwelling medical devices.[8]
It is now well documented that biofilms are notoriously
difficult to eradicate and are often resistant to systemic
102 Indian Journal of Medical Microbiology
antibiotic therapy and removal of infected device
becomes necessary.[3,9] According to National Institute
of Health, more than 60% of all infections are caused by
biofilm.[10] Biofilm organisms have an inherent resistance
to antibiotics, disinfectants and germicides. Unlike
planktonic populations, bacterial cells embedded in biofilms
exhibit intrinsic resistance to antibiotics due to several
specific defense mechanisms conferred by the biofilm
environment, including the inactivation of anti-microbial
agents by exopolysachharide (EPS), over expression of
stress-responsive genes, oxygen gradients within the
biofilm matrix and differentiation of a subpopulation of
biofilm cells into resistant dormant cells.[11,12] The intrinsic
resistance of bacterial cells within biofilms to conventional
anti-microbials has motivated new approaches for the
treatment of biofilm-associated infections, including
the use of silver preparations. Several silver-containing
dressings are recommended for long-term de-contamination
and wound healing based on silver’s broad-spectrum,
high-level anti-microbial activity.[13] The difficulty in
eradicating a chronic infection associated with biofilm
formation lies in the fact that biofilm bacteria are able
to resist higher antibiotic concentration than bacteria in
suspension.[14] Nanotechnology may provide the answer to
penetrate such biofilms and reduce biofilm formation. Silver
nanotechnology chemistry can prevent the formation of
life-threatening biofilms on medical devices. Silver is one
of the oldest known anti-microbials. It has recently been
demonstrated that AgNPs hydrogel hybrid with different
sizes of AgNPs can be effectively employed as anti-bacterial
agents.[15] Saxena et al. studied that propylene-based
sutures immobilised AgNPs show anti-bacterial activity
against S. aureus and Escherichia coli.[16] Due to the
strong anti-bacterial properties and low toxicity towards
mammalian cells, AgNPs have been applied in a wide range
of areas including wound dressing, coatings on medical
devices to reduce nosocomial infection rates,[17] protective
clothing, anti-bacterial surfaces, water treatment, food
preservation and cosmetics as biocidal and disinfecting
agents.[18] Although the literature reports that some studies
are related to the anti-bacterial activity of AgNPs, to the
authors’ knowledge, there are very few studies concerning
the effect of these particles against adhered cells and
biofilms of methicillin resistance S. aureus (MRSA)
and methicillin resistance S. epidermidis (MRSE).
Thus, the aim of the present study was to evaluate the
anti-biofilm potential of AgNPs against biofilms of
MRSA and MRSE by Congo Red Agar (CRA), scanning
electron microscopy (SEM) and confocal laser scanning
microscopy (CLSM).
Materials and Methods
Characterisation of silver nanoparticles
A stock solution of commercially available water
soluble AgNPs (5-10 nm) were procured from Nanoparticle
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vol. 33, No. 1
Biochem, Inc. (Columbia, USA). The subsequent dilutions
were made in autoclaved Milli Q water. The morphological
features and particle size of the procured NPs were
characterised by high-resolution transmission electron
microscopy (HR-TEM, Technai, FEI, Electron Optics,
USA) [Figure 1].
Bacterial strains, media and materials
A total of 62 non-repetitive clinical isolates of
Staphylococcus spp were recovered over a period of
6 months (September 2012 to February 2013) from skin
lesion such as pus, wounds, burn, etc., were subjected to the
study.
In vitro antibiotic susceptibility test
Individual isolates were tested, based on the
recommendations of the Clinical and Laboratory Standards
Institute (CLSI),[19] by the Kirby-Bauer disc diffusion
method for susceptibility to the following antibiotics:
Amikacin (AK, 30μg), Gentamycin (G, 10μg), Oxacillin
(Ox, 1 μg), Ceftriaxone (Ci, 30 μg), Cefotaxime (Ce,
30 μg), Vancomycin (Va, 30 μg), Chloramphenicol (C,
30 μg), Tobramycin (Tb, 10 μg), Novobiocin (No, 30
μg), Levofloxacin (Le, 5 μg), Clindamycin (Cd, 1 μg),
Erythromycin (E, 1 μg). Antibiotic discs used were procured
by Hi-Media (Mumbai, India).
Screening for methicillin resistance
Resistance to oxacillin was determined in all S. aureus
isolates using an oxacillin broth screening and disc diffusion
test recommended by the British Society for Antimicrobial
Chemotherapy (BSAC),[20] respectively. For the oxacillin
broth screening test, isolated colonies from an 18 to
24 h sheep blood agar plate were used to prepare a direct
inoculum equivalent to a 0.5 McFarland and suspended
in 2 ml nutrient broth, supplemented with 2% NaCl.
Breakpoints published by the CLSI were used: oxacillin
susceptible (S) <2 mg/L and resistant (R) ≥4 mg/L, on
Mueller-Hinton agar (MHA, Hi-Media, Mumbai, India)
plate with 2%NaCl incubated at 35°C for overnight.
For the disc diffusion test, a sterile swab was dipped in
the same S. aureus suspension as used for the oxacillin broth
Figure 1: HR-TEM image of AgNPs dispersion
January - March 2015 Ansari, et al.; Anti-biofilm efficacy of silver nanoparticles
screening test. The swab was plated on MHA plate in order
to get confluent growth (approx. 0.5 McFarland). Oxacillin
discs (1 μg) were applied on to the surface of inoculated
agar. Plates were incubated overnight at 30°C. An isolate
was classified as resistant to oxacillin when the inhibition
zone was ≤14 mm diameter.[20]
Detection of biofilm formation
Tissue culture plate method
The tissue culture plate (TCP) assay, described by
Christensen et al., is most widely used and was considered
as standard test for detection of biofilm formation.[21] A
total of 10 ml of Trypticase soy broth (TSB) with 1%
glucose was inoculated with a loopful of test organism from
overnight culture on nutrient agar. The broth was incubated
at 37°C for 24 h. The culture was further diluted 1:100 with
fresh medium. 96 wells flat bottom TCPs were filled with
0.2 ml of diluted cultures individually. Only sterile broth
was served as blank. Similarly control organisms were also
diluted and incubated. The culture plates were incubated at
37°C for 24 h. After incubation gentle tapping of the plates
was done. The wells were washed with 0.2 ml of phosphate
buffer saline (pH 7.2) four times to remove free floating
bacteria. Biofilms, which remained adherent to the walls
and the bottoms of the wells, were fixed with 2% sodium
acetate and stained with 0.1% crystal violet. Excess stain
was washed with de-ionised water and plates were dried
properly. Optical densities (OD) of stained adherent biofilm
were obtained with a micro ELISA autoreader at wave
length 570 nm. Experiment was performed in triplicate and
repeated thrice. Average of OD values of sterile medium
were calculated and subtracted from all test values.[25]
Tube method
A qualitative assessment of biofilm formation was
determined as previously described.[22] A total of 10 ml
TSB with 1% glucose was inoculated with a loopful of
microorganism from overnight culture plates and incubated
for 24 h at 37°C. The tubes were decanted and washed
with phosphate buffered saline (PBS) 0.1% (pH 7.3) and
dried. Dried tubes were stained with crystal violet (0.1%).
Excess stain was removed and tubes were washed with
de-ionised water. Tubes were than dried in inverted position
and observed for biofilm formation. Biofilm formation
was considered as positive, when a visible film lined the
wall and bottom of the tube. Ring formation at the liquid
interface was not indicative of biofilm formation.
Congo red agar method
Freeman et al. had described an alternative CRA
method for screening biofilm formation by Staphylococcus
isolates.[23] Plates were inoculated and incubated aerobically
for 24-48 h at 37°C. Positive result was indicated by
black colonies with a dry crystalline consistency. Weak
slime producers usually remained pink, though occasional
darkening at the centres of colonies was observed.
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103
A darkening of the colonies with the absence of a dry
crystalline colonial morphology indicated an indeterminate
result. The experiment was performed in triplicate and
repeated three times.
Scanning electron microscopy
Biofilms were assessed as previously described with or
without AgNPs. Briefly, the cells were washed with PBS,
fixed with 2.5% glutaraldehyde, then fixed samples were
subsequently washed again with PBS and dehydrated gently
by washing in a series of ethanol alcohol (30%, 50%, 70%,
80%, 95% and 100%) for 10 min at room temperature, and
critical point drying was performed. Afterwards, cells were
then oriented, mounted on the aluminium stubs and coated
with gold before imaging. The topographic features of the
biofilms were visualised with a SEM (Carl Zeiss EVO 40,
Germany) with accelerating voltage of 20 kV.
Confocal laser scanning microscopy
Biofilms for confocal analysis were grown on glass
coverslips as previously described.[24] Briefly, 12-well
microtitre plate seeded with glass coverslips were incubated
for 24 h at 37°C in 5 ml of brain heart infusion (BHI) with
5% sucrose. The wells were inoculated with 100 μl of
mid-exponential grown culture of S. aureus After 24 h, the
cover slips were removed and gently washed three times with
sterile PBS and were first stained with 15 μM Propidium
iodide for 5 min at room temperature to detect bacterial
cells in red. After being washed in PBS, the sections were
incubated with 50 μg/ml of concanavalin A-fluorescein
isothiocyanate (Con A-FITC) (C7642; Sigma-Aldrich
Inc, St Louis, MO, USA) for 5 min at room temperature to
stain the glycocalyx matrix green. The Propidium iodide
was excited at 520 nm, the emission was monitored at
620 nm and Con A-FITC was excited and monitored at 495
and 525 nm, respectively. Intact biofilms were examined
non-destructively using a Fluoview FV1000 Espectral
Olympus CSLM (Olympus Latin America, Miami, FL, USA)
equipped with a UPlanSApo 100X/1.40 oil UIS2 Olympus oil
immersion lens. Optical sections of 0.87 μm were collected
from the complete thickness of the biofilms. Then, for each
sample, images from three randomly selected positions were
obtained and analysed using an Olympus Fluoview FV 1000.
Results
Prevalence of bacterial isolates
Sixty-two Staphylococcus species were isolated from
the patients associated generally with the diseases of
purulent dermatitis, burns, wounds and other localised
skin infections. Out of 62 isolates screened, 25 (43.9%)
isolates were MRSA, 27 (47.5%) were methicillin sensitive
S. aureus (MSSA), 6 (5.3%) were methicillin resistance
Coagulase negative Staphylococcus (MR-CONS) and
4 (3.5%) methicillin sensitive Coagulase negative
Staphylococcus (MS-CONS).
104 Indian Journal of Medical Microbiology vol. 33, No. 1

Antibiotic susceptibility testing
Results obtained from investigation of resistance in
Staphylococcus spp isolates, which were susceptible or
resistant to individual antibiotics, are displayed in Table 1.
Evaluation of anti-bacterial activity of AgNPs
The MIC and MBC of AgNPs dispersion tested against
MRSA, MSSA and MRSE were observed in the range of
11.25-45 μg/ml [Table 2a and b]. The result clearly shows
that AgNPs exhibit excellent bacteriostatic and bactericidal
effect regardless of their drug-resistant mechanisms.
Screening of biofilm formation 0n CRA
Table 3a shows that out of 52 isolates of S. aureus and
10 of S. epidermidis tested for biofilm formation by CRA
method, 41 (78.85%) isolates of S. aureus and 8 (80%)
isolates of S. epidermidis produced black colonies.
However, only 25 (48.08%) isolates of S. aureus and
5 (50%) isolates of S. epidermidis colonies were black in
colour with dry crystalline consistency, which is indicative
of biofilm formation. A total of 16 (30.77%) S. aureus
and 3 (30%) S. epidermidis isolates were black in colour
but were not dry and crystalline and were indeterminate
for biofilm formation. These isolates were also taken as
negative for biofilm formation. A total of 11 (21.15%)
S. aureus and 2 (20%) S. epidermidis isolates produced
pink colonies, which were taken as negative for biofilm
formation.
Screening of biofilm formation by TCP method
In the quantitative assay for the biofilm production,
the isolates were classified as highly biofilm
producing (strongly adherent), moderate adherent
isolates and non-biofilm producers (weak/non-adherent).
Quantitative microtitre assay for biofilm formation was
strongly positive in six (11.54%) isolates of S. aureus
and three (30%) S. epidermidis, while the remaining
isolates were either moderate adherent 30 (57.69%) S.
aureus and 4 (40%) S. epidermidis or weak/non-biofilm
producers 16 (30.77%) S. aureus and 3 (30%) S.
epidermidis [Table 3b]. Isolates screened for biofilm
formation by microtitre plate assay method are shown in
Figure 2.

Screening of biofilm formation by tube method

Table 1: Antimicrobial susceptibility pattern of
Staphylococcus species (N=62) Qualitative tube method of biofilm screening of bacterial
Antibiotics S. aureus isolates CONS isolates showed that 33 (63.46%) isolates of S. aureus and
(μg) (n=52) (%) (n=10) (%) 7 (70%) isolates of S. epidermidis were positive for biofilm
MRSA MSSA MR MS production [Table 3c]. Most of the isolates showed thick
(n=25) (n=27) (n=6) (n=4) blue ring at the liquid-air interface.
Amikacin 19 (76) 27 (100) 2 (33.3) 4 (100) Characterisation of anti-biofilm activity of AgNPs on CRA
Gentamycin 19 (76) 27 (100) 4 (66.6) 4 (100) Plates
Oxacillin 0 (0) 27 (100) 0 (0) 4 (100)
Vancomycin 25 (100) 27 (100) 6 (100) 4 (100) Biofilm formation is detected in many organisms
Novobiocin 25 (100) 27 (100) 0 (0) 0 (0) synthesising exopolysachharides. The biofilm is made up
Levofloxacin 18 (72) 27 (100) 2 (33.3) 4 (100) of microorganisms adhering to the surface coated with
Clindamycin 16 (64) 26 (96.2) 2 (33.3) 4 (100) slime - the exopolysachharide matrix which protects the
Erythromycin 12 (48) 27 (100) 4 (66.6) 2 (50) microbes from the unfavourable environmental factors.
MRSA: Methicillin resistance S. aureus, MSSA: Methicillin sensitive Biofilm formation by S. aureus isolates were tested
S. aureus, MR: Methicillin resistance, MS: Methicillin sensitive by growing the organism in brain-heart infusion agar

Table 2: MIC and MBC values of AgNPs tested against clinical isolates of Staphylococcus species
MRSA 25 (48.1%) MSSA 27 (51.9%)
Isolates (%) MIC μg/ml Isolates (%) MBC μg/ml Isolates (%) MIC μg/ml Isolates (%) MBC μg/ml
48 11.25 72 22.50 51.85 11.25 77.78 22.50
32 22.50 28 45.00 33.33 22.50 22.22 45.00
20 45.00 - - 14.81 45.00 - -
MRSE 6 (60%) MSSE 4 (40%)
Isolates (%) MIC μg/ml Isolates (%) MBC μg/ml Isolates (%) MIC μg/ml Isolates (%) MBC μg/ml
16.66 11.25 16.67 22.50 75 22.50 100 45.00
66.66 22.50 83.33 45.00 25 45.00 - -
16.66 45.00 - - - - - -
MIC: Minimum inhibitory concentration, MRSA: Methicillin resistant S. aureus, MARE: Methicillin resistant S. epidermidis,
MSSA: Methicillin sensitive S. aureus, MBC: Minimum bactericidal concentration, MSSE: Methicillin sensitive S. epidermidis

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January - March 2015 Ansari, et al.; Anti-biofilm efficacy of silver nanoparticles 105

Table 3: Screening of biofilm formation of

Staphylococcus species by (a) Congo Red Agar,
(b) Tissue Culture Plate and (c) Tube methods
S. aureus S. epidermidis
(%) (%)
(a) Colony appearance on (CRA)
Pink/Red (negative) 11 (21.15) 2 (20)
Black colonies without 16 (30.77) 3 (30)
dry crystalline consistency
(indeterminate)
Black colonies with dry 25 (48.08) 5 (50)
crystalline consistency (positive) Figure 2: Screening of biofilm producers by TCP method: High,
(b) Biofilm production (TCP) moderate and non-slime producers differentiated with crystal violet
staining were shown on 96 well tissue culture plates
Weak/Non (<0.120 OD) 16 (30.77) 3 (30)
Moderate (0.12-0.24 OD) 30 (57.69) 4 (40)
High (>0.24 OD) 6 (11.54) 3 (30)
(c) Biofilm Production (TM)
0/1 (weak/non ) 19 (36.54) 3 (30)
2+ (moderate) 25 (48.08) 4 (40)
3+ (strong) 8 (15.39) 3 (30)
CRA: Congo Red Agar, TCP: Tissue culture plate, TM: Tube
methods a c

supplemented with Congo Red (BHIC) with and without

silver nanoparticles. When the colonies were grown
without AgNPs in the medium, the organisms appeared as
dry crystalline black colonies, indicating the production
of exopolysachharides, which is the prerequisite for
the formation of biofilm [Figure 3]. Whereas when
the organisms were grown on BHIC with AgNPs, the b d
organisms did not survive. During the treatment with Figure 3: Ability of the organisms was checked for biofilm formation
reduced concentrations of AgNPs (10 μg/ml), the organisms in BHI agar supplemented with Congo Red. The appearance of
black crystalline colonies (a and b) indicate the exopolysachharide
continued to grow, but AgNPs treatment has inhibited the production by MRSA (a) and MRSE (b), whereas the addition of
synthesis of glycocalyx matrix, indicated by the absence of 50 μg/ml AgNPs blocked the exopolysachharide synthesis by MRSA
dry crystalline black colonies [Figure 3]. It was found that (c) and MRSE (d) and also inhibited the growth of the organism itself
at higher concentration of AgNPs inhibited bacterial growth
by more than 98%. When the glycocalyx matrix synthesis
is arrested, the organism cannot form biofilm. It was also
observed that 20 μg/ml of AgNPs significantly arrested
biofilm formation without affecting viability, whereas
50 μg/ml completely blocked the biofilm formation and
inhibited the growth of the organism itself.

Characterisation of anti-biofilm activity of AgNPs by

scanning electron microscopy

The biofilm grown on glass slide for 24 h was observed a b

using SEM. The biofilm formed by the native strain have
aggregated and clumped bacterial cells [Figure 4a]. The
S. aureus cultures grown without AgNPs exhibit the
expected normal cellular morphology with smooth cell
surfaces [Figure 4a]. Under the same growth conditions
but in the presence of AgNPs (20 μg/ml) S. aureus
cell shows changes in morphology and it was also
examined that AgNPs inhibit bacterial colonisation on
the surfaces [Figure 4b]. The apparent biofilm formed by
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Figure 4: SEM images of S. aureus biofilms developed on the glass
slide surface at 24 h of incubation. Untreated (a) and treated (b) with
AgNPs (20 μg/ml)
S. aureus has very few cells individually scattered along
the surface. The cells were arranged in short chains with
absence of exopolysachharides matrix. The biofilm formed
by the S. aureus was very much patchy [Figure 4a]. More
specifically, an obvious increase in the roughness of the
106 Indian Journal of Medical Microbiology vol. 33, No. 1

cell surface suggested that it has been damaged by the
nanoparticles. Microscope evaluation of the surfaces clearly
shows that the AgNPs treated glass surfaces do not allow
bacterial colonisation and biofilm formation compared with
the untreated controls. Untreated glass surfaces supported a
massive biofilm formation by S. aureus [Figure 4a], while
AgNPs treated glass surfaces shows dramatically restricted
bacterial colonisation and biofilm formation [Figure 4b].
These results suggest that AgNPs are effective in restraining
bacterial colonisation of the surface.
Characterisation of anti-biofilm activity of AgNPs by
Confocal laser scanning microscopy
CLSM analysis of biofilms formation by
MRSA [Figure 5a] and MRSE [Figure 5b] was performed
to examine the effects of coating of AgNPs on biofilm
architecture using AgNPs-coated glass cover slips in a
12-well microtitre plate. To visualise bacterial cells and
the surrounding glycocalyx matrix (which is indicative
of bacterial biofilm formation), double staining was
performed using propidium iodide and Con A-FITC.
Bacterial cells stained red and were easily identified by
their size and morphologic features, when using propidium
iodide. Whereas Con A-FITC binds to mannose residues
resulting in green staining and indicating the presence
of a bacterial glycocalyx. Although, we observed the
marked co-localisation of green Con A-FITC staining
with clusters of bacterial cells, the staining of the matrix
was not homogeneously distributed. The presence of
dark areas within the biofilm can be explained by the
existing water channels, the heterogeneous production
of the matrix and the types of exopolysachharides
within the biofilm, as well as the absence of Con A-FITC
binding to the matrix. When CLSM images with red and
green fluorescent intensities were superimposed, yellow
colour (green + red) revealed that the extracellular-Con
A-FITC-reactive polysaccharide (green) was produced
in the intra-cellular spaces (red), indicating thereby that
extracellular polysaccharides were produced as a capsular
component in biofilm. We observed that interconnected
bacteria were encased in a scaffolding network composed of

b
Figure 5: CLSM micrographs of MRSA (a) and MRSE (b) biofilm. Panel 1 from left to right represents CLSM images of native biofilm (without
AgNPs). Whereas panel 2 from left to right represent CLSM images of MRSA (a) and MRSE (b) biofilm treated with AgNPs and most of the cells
exhibited shape distortions and slight elongation and almost no exopolysachharides (green fluorescent) was observed. The number of live bacterial
cells was reduced significantly, and the 3-dimensional structure was also disrupted. Magnification ×100
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January - March 2015 Ansari, et al.; Anti-biofilm efficacy of silver nanoparticles
extracellular matrix, suggesting a 3-dimensional architecture
of biofilm formations. The micrographs suggest that the
biofilm formed on uncoated AgNPs surfaces covered a
larger surface area and had a definite architecture (Figure 5a
and b, first panel). However, the biofilms formed on
coverslips coated with AgNPs showed no or few spread
cells with no distinct pattern of arrangement. S. aureus
cells exhibited shape distortions, indentations and slight
elongation (Figure 5a and b second panel), after exposure
to AgNPs. The cells grown in the presence of 25 μg/ml
AgNPs show a complete absence of clumped cells and
were individually scattered over the surface rather than in
any arrangement. Uncoated cover slips surfaces supported
a massive biofilm formation of all bacterial cells, whereas
AgNPs-coated surfaces dramatically restricted bacterial
colonisation.
Discussion
In recent years, there has been considerable interest
in the problems posed by the biofilm mode of bacterial
and fungal growth. According to public announcement
from national institute of health, more than 60% of all
microbial infection is caused by biofilms.[25] Infections
resulting from microbial biofilm formation remain a serious
threat to patients worldwide. Particularly problematic are
wound infections,[26] with chronic wounds such as foot,
leg and pressure ulcers being particularly susceptible to
biofilm infections.[27] In order to kill or remove biofilms,
anti-microbials must penetrate the polysaccharide matrix
to gain access to the microbial cells. Nanotechnology may
provide the answer to penetrate such biofilms and reduce
biofilm formation by the use of ‘nano functionalisation’
surface techniques to prevent the biofilm formation.
Biofilm formation by clinical isolates of S. aureus was
tested by CRA, tube and TCP methods. However, the
anti-biofilm efficacy of AgNPs was investigated by growing
the organism on CRA supplemented with and without
AgNPs. When the colonies were grown without AgNPs,
the organisms appeared as dry crystalline black colonies,
indicating the production of exopolysachharides, which
is the prerequisite for the formation of biofilm [Figure 3].
Whereas when the organisms were grown with AgNPs,
the organisms did not survive. During the treatment with
reduced concentrations of AgNPs (10 μg/ml), the organisms
continued to grow, but AgNPs treatment has inhibited the
synthesis of glycocalyx matrix, indicated by the absence
of dry crystalline black colonies. However, at higher
concentration of AgNPs (50 μg/ml) almost no growth was
observed [Figure 3]. Thus, when the glycocalyx synthesis
is arrested, the organism cannot form biofilm. Similar
results were also reported by Kalishwaralal et al. against
P. aeruginosa and S. epidermidis biofilms and found
that 100 nM of AgNPs resulted in a 95-98% reduction in
biofilm.[28] It was also cited in literature that if the surface of
medical devices has an AgNPs coating, then it was helpful
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107
in preventing bacterial adhesion and subsequent biofilm
formation on the medical devices.[29]
Based on these results we continued to address the
mechanism of action of AgNPs on biofilm forming
bacterial species by applying CLSM and SEM. SEM was
used to examine cell morphologies following exposure
to the nanoparticles. S. aureus cultures grown without
nanoparticles exhibit the expected normal cellular
morphology with smooth cell surfaces [Figure 4a]. Under
the same growth conditions but in the presence of suspended
AgNPs (30 μg/ml), S. aureus bacteria presented a change
in morphology [Figure 4b]. More specifically, an obvious
increase in the roughness of the cell surface suggested
that it has been damaged by the nanoparticles. EPS
within S. aureus biofilms was not detected by SEM. SEM
observations clearly indicate that AgNPs reduced the surface
coverage by S. aureus [Figure 4b] biofilms. Our results
are in agreement with previously reported anti-biofilm
activity of nanocrystalline silver by Kostenko et al.[30] They
observed that nanocrystalline silver dramatically decreased
viable cell numbers within the tested biofilms. SEM results
shows that nanocrystalline silver reduced the surface
coverage by P. aeruginosa biofilms. Prolonged treatment
with nanocrystalline silver provided a further reduction
in the surface coverage by MRSA biofilms. The surface
coverage by E. coli biofilms was reduced by the tested
dressings by approximately 20% after the first day.[30]
In order to achieve a fundamental understanding of the
formation and presence of bacterial biofilms, the analysis
should include detection of the bacteria and the matrix. The
most common methods of assessing biofilm heterogeneity
are direct microscopic imaging of local biofilm morphology
or microscopic measurement of the local biofilm thickness.
For many applications, time lapse microscopy using CLSM
is an ideal tool for monitoring at a spatial resolution of
the order of micrometers, and allows the non-destructive
study of biofilms through an examination of all the layers
at different depths, thus making it is possible to reconstruct
a 3-dimensional structure.[31] The detection of the matrix
can be achieved using a double-staining technique in
combination with CLSM, which allows the simultaneous
imaging of bacterial cells as well as glycocalyx within
biofilms.[32]
Therefore, to visualise the bacterial cells and the
surrounding glycocalyx matrix (which is indicative
of bacterial biofilm formation), we used this powerful
technique (CLSM) to examine the effect of nanoparticles
on glycocalyx matrix/exopolysachharides synthesis; double
staining was performed using propidium iodide and Con
A-FITC. In case of untreated S. aureus (Figure 5a and b,
panel 1), PI stain bacterial nucleic acids and fluorescent
red, while, green fluorescent (Con A-FITC) around bacteria
indicates the presence of exopolysachharides. Whereas
108 Indian Journal of Medical Microbiology
AgNPs treated samples of S. aureus (Figure 5a and b,
panel 2) shows that most of the cells were dead and no
glycocalyx matrix (green fluorescent) was observed. The
number of live bacterial cells was reduced significantly,
and the 3-dimensional structure was also disrupted. We
investigated that this inhibitory effect of AgNPs on the
existing biofilm may due to be presence of water channels
though out the biofilm. Since in all biofilms, water
channels (pores) are present for nutrient transportation,
AgNPs may directly diffuse through the glycocalyx matrix
layer through the pores and may impart anti-microbial
function. Masurkar et al. investigated the anti-biofilm
activity of AgNPs (51 nm) synthesised from B. megaterium
and reported that AgNPs showed enhanced quorum
quenching activity against S. aureus biofilm and prevention
of biofilm formation, which can be seen under inverted
microscope. They concluded that AgNPs might be involved
in neutralising these adhesive substances, thus preventing
biofilm formation.[33]
However, the exact mechanism of action of AgNPs in
biofilm-related studies is yet to be demonstrated. Kostenko
et al. reported that Acticoat nanocrystalline silver has the
highest anti-biofilm efficacy compared with Aquacel silver
and Silverlon and the silver concentration alone cannot
account for the anti-biofilm efficacy of the silver dressings.
The type of silver species present also plays a role.[30]
The reduction of the silver particle to the nanoscale level
increases the relative surface area, which provides higher
Ag+ release rates than for elemental silver particles.[34]
Moreover, nanoparticles have a higher capacity to attach to
and penetrate bacterial membranes and accumulate inside
cells, providing a continuous release of silver ions inside the
cell.[35-37]
Conclusion
When MRSA and MRSE assume the biofilm phenotype,
these infections are often extremely difficult to treat. The
infection may fail to respond to antibiotic therapy or it may
initially respond only to relapse weeks or months later. In
such cases, invasive treatments, such as surgical removal
and replacement of the infected tissue or device, may be
required. So for proper treatment of S. aureus infection
screening for biofilm production is necessary. The presence
of biofilms on medical devices or surfaces can only be
observed using a limited number of techniques. The reason
why the demonstration of bacterial biofilms is challenging
is because it is difficult to stain both the bacteria and
glycocalyx. Furthermore, light and electron microscopy
techniques require a dehydration process that reduces the
total volume of the matrix and alters its architecture. CLSM
is an ideal tool for monitoring at a spatial resolution of
the order of micrometers, and allows the non-destructive
study of biofilms through an examination of all the layers
at different depths, thus making it possible to reconstruct
a 3-dimensional structure. The detection of the glycocalyx
www.ijmm.org
vol. 33, No. 1
matrix can be achieved using a double-staining technique
in combination with CLSM, which allows the simultaneous
imaging of bacterial cells as well as glycocalyx within
biofilms. In the near future, the AgNPs may play major role
in the coating of medical devices and treatment of infections
caused due to highly antibiotic-resistant biofilm.
Acknowledgement
The authors would like to thank the Indian Council of Medical
Research (ICMR) New Delhi-India, grant number 35/15/BMS-11
for their partial support and funding of this project.
References
1. Donlan RM. Biofilms and device associated infections. Emerg
Infect Dis 2001;7:277-81.
2. Kloos WE, Bannerman TL. Update on clinical significance
of coagulase-negative Staphylococci. Clin Microbiol Rev
1994;7:117-40.
3. Schwank S, Rajacic Z, Zimmerli W, Blaser J. Impact of
bacterial biofilm formation on in vitro and in vivo activities of
antibiotics. Antimicrob Agents Chemother 1998;42:895-8.
4. Rhoads DD, Wolcott RD, Percival SL. Biofilms in wounds:
Management strategies. J Wound Care 2008;17:502-8.
5. O’Toole G, Kaplan HB, Kolter R. Biofilm formation as
microbial development. Annu Rev Microbiol 2000;54:49-79.
6. O’Gara JP, Humphreys H. Staphylococcus epidermidis
biofilms: Importance and implications. J Med Microbiol
2001;50:582-7.
7. Ammendolia MG, Rosa RD, Montanaro L, Arciola CR,
Baldassarri L. Slime production and expression of
slim-associated antigen by staphylococcal clinical isolates.
J Clin Microbiol 1999;37:3235-8.
8. Arciola CR, Baldassarri L, Montanaro L. Presence of icaA
and icaD genes and slime production in a collection of
staphylococcal strains from catheter-associated infections.
J Clin Microbiol 2001;39:2151-6.
9. Souli M, Giamarellou H. Effects of Slime produced by clinical
isolates of coagulase negative staphylococci on activities of
various antimicrobial agents. Antimicrob Agents Chemother
1998;42:939-41.
10. Kim L. Riddle of biofilm resistance. Antimicrob Agents
Chemother 2001;45:999-1007.
11. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister
cells and tolerance to antimicrobials. FEMS Microbiol Lett
2004;230:13-8.
12. Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival
strategies of infectious biofilms. Trends Microbiol
2005;13:34-40.
13. Leaper DJ. Silver dressings: Their role in wound management.
Int Wound J 2006;3:282-94.
14. Gristina AG, Hobgood CD, Web LX, Myrvik QN. Adhesive
colonization of biomaterials and antibiotics resistance.
Biomaterials 1987;8:423-6.
15. Mohan YM, Lee K, Premkumar T, Geckeler KE. Hydrogel
networks as nonreactors: A novel approach to silver
nanoparticles for antibacterial applications. Polymer
2007;48:158-64.
16. Saxena S, Ray AR, Kapil A, Pavon-Djavid G, Letourneur D,
Gupta B, et al. Development of a new polypropylene-based
suture: Plasma grafting, surface treatment, characterization,
January - March 2015 Ansari, et al.; Anti-biofilm efficacy of silver nanoparticles
and biocompatibility studies. Macromol Biosci 2011;
11:373-82.
17. Chaloupka K, Malam Y, Seifalian AM. Nanosilver as a new
generation of nanoproduct in biomedical applications Trends
Biotechnol 2010;28:580-8.
18. Moritz M, Geszke-Moritz M. The newest achievements
in synthesis, immobilization and practical applications of
antibacterial nanoparticles. Chem Engg J 2013;228:596-613.
19. CLSI. Performance standards for antimicrobial susceptibility
testing, Fifteenth Informational Supplement, CLSI document
2006; M100-S16, vol. 26-3; M7-A7, vol. 26-2; M2-A9,
vol. 26-1. Wayne, PA, USA.
20. BSAC (British Society for Antimicrobial Chemotherapy).
Methods for antimicrobial susceptibility testing.
Version 10.2 May 2011.
21. Christensen GD, Simpson WA, Younger JA, Baddour LM,
Barrett FF, Melton DM, et al. Adherence of coagulase
negative Staphylococci to plastic tissue cultures:
A quantitative model for the adherence of staphylococci to
medical devices. J Clin Microbiol 1985;22:996-1006.
22. Christensen GD, Simpson WA, Bisno AL, Beachey EH.
Adherence of slime-producing strains of Staphylococcus
epidermidis to smooth surfaces. Infect Immun 1982;
37:318-26.
23. Freeman DJ, Falkiner FR, Keane CT. New method
for detecting slime production by coagulase negative
staphylococci. J Clin Pathol 1989;42:872-74.
24. Banas JA, Hazlett KR, Mazurkiewicz JE. An in
vitro model for studying the contributions of the
Streptococcus mutans glucan-binding protein-A to biofilm
structure. Methods Enzymol 2001;337:425-33.
25. Lewis K. Riddle of biofilm resistance. Antimicrob Agents
chemother 2001;45:999-1007.
26. Percival SL, Bowler P, Woods EJ. Assessing the effect of an
antimicrobial wound dressing on biofilms. Wound Repair
Regen 2008;16:52-7.
27. James GA, Swogger E, Wolcott R, deLancey Pulcini E, Secor
P, Sestrich J, et al. Biofilms in chronic wounds. Wound Repair
Regen 2010;16:37-44.
28. Kalishwaralal K, BarathManiKanth S, Pandian SR,
Deepak V, Gurunathan S. Silver nanoparticles impede
the biofilm formation by Pseudomonas aeruginosa and
Staphylococcus epidermidis. Colloids Surf B Biointerfaces
2010;79:340-4.
29. Knetsch ML, Leo HK. New strategies in the development of
www.ijmm.org
109
antimicrobial coatings: The example of increasing usage of
silver and silver nanoparticles. Polymers 2011;3:340-66.
30. Kostenko V, Lyczak J, Turner K, Martinuzzi TJ. Impact
of silver-containing wound dressings on bacterial
biofilm viability and susceptibility to antibiotics during
prolonged treatment. Antimicrob Agents Chemother 2010;
54:5120-31.
31. Lawrence JR, Neu TR. Confocal laser scanning microscopy
for analysis of microbial biofilms. Methods Enzymol
1999;310:131-44.
32. Kania RE, Lamers GE, Vonk MJ, Huy PT, Hiemstra PS,
Bloemberg GV, et al. Demonstration of bacterial cells and
glycocalyx in biofilms on human tonsils. Arch Otolaryngol
Head Neck Surg 2007;133:115-21.
33. Masurkar SA, Chaudhari PR, Shidore VB, Kamble SP.
Effect of biologically synthesized silver nanoparticles on
Staphylococcus aureus biofilm quenching and prevention of
biofilm formation. IET Nanobiotechnol 2012;6:110-4.
34. Dunn K, Edwards-Jones V. The role of Acticoat with
nanocrystalline silver in the management of burns. Burns
2004;30:1-9.
35. Rai M, Yadav A, Gade A. Silver nanoparticles as a new
generation of antimicrobials. Biotechnol Adv 2009;27:76-83.
36. Ansari MA, Khan HM, Khan AA, Cameotra SS, Pal R.
Antibiofilm efficacy of silver nanoparticles against biofilm of
extended expectrum β-lactamase isolates of Escherichia coli
and Klebsiella pneumoniae. Appl Nanosci 2013. [In press].
DOI: 10.1007/s13204-013-0266-1.
37. Ansari MA, Khan HM, Khan AA, Cameotra SS, Saquib Q,
Musarrat J. Gum arabic capped-silver nanoparticles inhibit
biofilm formation by multi-drug resistant strains of
Pseudomonas aeruginosa. J Basic Microbiol 2014; 54:1-12.
How to cite this article: Ansari MA, Khan HM, Khan AA, Cameotra SS,
Alzohairy MA. Anti-biofi lm efficacy of silver nanoparticles against
MRSA and MRSE isolated from wounds in a tertiary care hospital.
Indian J Med Microbiol 2015;33:101-9
Source of Support: The authors would like to acknowledge SAIF-
DST, Department of Anatomy, All India Institute of Medical Sciences
(AIIMS), New Delhi, India, for HR-TEM and Advanced Instrumentation
Research facility (AIRF), Jawaharlal Nehru University, New Delhi,
India for SEM and Confocal laser scanning electron microscopy
observation of AgNPs nanoparticle and bacterial interaction,
Conflict of Interest: None declared.
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